REVIEW: Role of Soluble Guanylate Cyclase in the Molecular Mechanism
Underlying the Physiological Effects of Nitric Oxide

I. S. Severina

Received June 26, 1997
In this review the molecular mechanisms underlying the antihypertensive
and antiaggregatory actions of nitric oxide (NO) are discussed. It has
been shown that these effects are directly connected with the
activation of soluble guanylate cyclase and the accumulation of cyclic
3´,5´-guanosine monophosphate (cGMP). The mechanism of
guanylate cyclase activation by NO is analyzed, especially the role and
biological significance of the nitrosyl--heme complex formed as a
result of interaction of guanylate cyclase heme with NO and the role of
sulfhydryl groups of the enzyme in this process. Using new approaches
for studying the antihypertensive and antiaggregatory actions of nitric
oxide in combination with the newly obtained data on the regulatory
role of guanylate cyclase in the platelet aggregation process, the most
important results were obtained regarding the molecular bases providing
for a directed search for and creation of new effective
antihypertensive and antiaggregatory preparations. In studying the
molecular mechanism for directed activation of soluble guanylate
cyclase by new NO donors, a series of hitherto unknown enzyme
activators generating NO and involved in the regulation of hemostasis
and vascular tone were revealed.
KEY WORDS: guanylate cyclase, nitric oxide, platelet aggregation

Determination of the endogenous nature of nitric oxide (NO) was one of
the most fundamental achievements in biochemistry of recent years; it
allows better insight into the molecular mechanisms underlying several
physiological processes in cells. Nitric oxide proved to be identical
to endothelium-derived relaxing factor (EDRF) [1].

Endogenous NO is formed from L-arginine by oxidation of the guanidine
amine group with catalysis by L-arginine-NO-synthase [2]. Endogenous NO is known to be involved in several
important physiological processes, acting as a neurotransmitter [3], a cytotoxic agent [4], and a
powerful factor in hemostasis. NO inhibits platelet aggregation [5] and is presently considered as the endogenous
vasodilator. Antihypertensive and antiaggragatory properties of nitric
oxide are directly associated with the functions of guanylate
cyclase.

Guanylate cyclase (EC 4.6.1.2) catalyzes the biosynthesis of cyclic
3´,5´-guanosine monophosphate (cGMP) from guanosine
triphosphate. cGMP is a potent regulator of cell metabolism. The
hydrolytic degradation of cGMP is catalyzed by phosphodiesterase.
However, the key enzyme of cGMP metabolism is guanylate cyclase, and it
is due to activation of this enzyme that the increase in tissue cGMP
level is brought about.

Guanylate cyclase exists in two forms: soluble and membrane bound. It
has been established that these forms are not only two different
proteins, but two differently regulated enzymes [6]. The present paper discusses the soluble form of
guanylate cyclase because it is with this enzyme the antihypertensive
and antiaggregatory action of nitric oxide is associated. Soluble
guanylate cyclase is a heterodimer with two immunologically distinct
subunits. One characteristic feature of the enzyme is the presence in
its molecule of labile sulfhydryl groups. Oxidation of the latter
stimulates the enzyme activity [7]. However,
long-term exposure of the enzyme to oxidants results in the loss of
activity [8]. The second characteristic feature of
soluble guanylate cyclase is the presence of heme in the enzyme
molecule [9]. It is known that the immediate heme
precursor in vivo is protoporphyrin IX, which proved to be a
potent activator of the enzyme [10]. Introduction
of iron in the porphyrin ring yields ferroprotoporphyrin IX (or heme)
which acts as an inhibitor of the enzyme [11]. The
sequence of reactions resulting in the formation of the guanylate
cyclase holoenzyme, namely, protoporphyrin IX -- iron -- heme --
guanylate cyclase, remains obscure. However, this system is essential
to the endogenous regulation of guanylate cyclase.

The role of heme in the functioning of guanylate cyclase is thought to
be mainly connected with enzyme activation by nitric oxide and
NO-generating compounds [12, 13]. The actual activator of the enzyme is the
nitrosyl--heme complex [14] which is formed on
interaction of a NO group with the guanylate cyclase heme. Under these
conditions the iron protrudes from the plane of the porphyrin ring; as
a result the structure of the nitrosyl--heme complex formed becomes
similar to that of protoporphyrin IX--one of the potent activators of
the enzyme [11]. It was found that heme-deficient
guanylate cyclase loses its capacity to be activated by NO [14]. On reconstruction of the heme-deficient enzyme
(by hematin in the presence of dithiothreitol) to the heme-containing
guanylate cyclase, the enzyme recovers its sensitivity to activation by
NO and NO-generating compounds [15].
Nitrosyl--heme complexes may be formed by interactions between NO or
NO-donors with heme moieties of other heme-containing proteins
(hemoglobin, myoglobin, catalase, etc.). Guanylate cyclase has a high
affinity to the nitrosyl--heme complex, facilitating its transfer from
other heme-containing proteins to the enzyme but not in the opposite
direction [16].

The heme-dependent activation of the enzyme was proposed to be related
to its sulfhydryl groups because the capacity of the enzyme to be
activated by NO decreases after oxidation [17].
Another line of evidence comes from the finding that heme-deficient
enzyme is still sensitive to activation by NO and NO-generating
compounds, though their efficacy falls considerably [18, 19].

For the last few years (1994-1997) new data on the functional domains of
soluble guanylate cyclase and the role of the cysteine residues of the
enzyme in its functioning were obtained. It was shown that soluble
guanylate cyclase heterodimer consists of two subunits: alpha
and beta [20]. Thus the first evidence was
provided that the regulatory and catalytic properties of guanylate
cyclase can be attributed to different regions of the subunits and that
the catalytic site is located in the COOH-terminal halves of the
alpha- and beta-subunits of guanylate cyclase [21]. This catalytic site is responsible only for cGMP
formation and is insensitive to NO. The NH2-terminal region
of the beta-subunit is involved in activation of guanylate
cyclase by nitric oxide [21, 22]. The site of heme binding is still unknown, but
point mutation of His-105 to Phe in the beta-subunit disrupts
heme binding with the protein and the resulting recombinant guanylate
cyclase loses its ability to be activated by NO. At the same time, the
basal catalytic guanylate cyclase activity is unchanged [21, 22]. Point mutation of 15
conserved cysteine residues located in the alpha- and
beta-subunits of guanylate cyclase to serine yielded recombinant
enzymes that were able to catalyze the synthesis of cGMP. Mutation of
two cysteine residues (Cys-78 and Cys-214) located in the immediate
proximity of His-105, the putative heme-binding region of the
beta-subunit, yielded recombinant proteins that were insensitive
to NO [23]. The role of SH-groups of soluble
guanylate cyclase in enzyme activation by nitric oxide is not yet
clear. It was proposed that the interaction between thiols and NO and
NO-generating compounds produces S-nitrosothiols [24], which had been earlier considered the true
activators of guanylate cyclase [24]. However, now
S-nitrosothiols are regarded as intermediate NO donors rather than the
direct stimulant of guanylate cyclase [16].
Indeed, thiols facilitate activation of guanylate cyclase by NO donors
[25]. There is no consensus in the literature on
the role of sulfhydryl groups of the enzyme in its activation by NO and
NO donors. Apparently, the stimulatory effect may be complex and
depends on the presence of thiols in the sample and on the degree of
oxidation of guanylate cyclase. However, a major role is played by the
guanylate cyclase heme and the nitrosyl--heme complex; the latter is
the true activator of the enzyme.

MECHANISM UNDERLYING THE ANTIHYPERTENSIVE ACTION OF NITRIC
OXIDE

Apparently, the antihypertensive action of nitric oxide is directly
connected with the heme dependent mechanism of guanylate cyclase
activation and accumulation of cGMP. The accumulated cGMP activates
cGMP-dependent protein kinase and Ca2+-ATPase, which
dephosphorylate myosin light chain leading to the efflux of
Ca2+ from muscle cells and ultimately to vascular relaxation
[26]. The therapeutic effects of most known
nitrovasodilaters (such as glycerotrinitrate, nitrosorbide, etc.) are
closely related to the interaction of NO released in the course of
their biotransformation with the guanylate cyclase heme (according to
the above mechanism), enzyme activation, and accumulation of cGMP.

The nature of the bond between the protein and heme in guanylate cyclase
has not yet been determined; however, the lability of this bond has
been proved. The heme can dissociate from the protein on a decrease in
pH (5.0), on storage, or purification of the enzyme, thus providing a
certain degree of heme deficiency of guanylate cyclase. The tightness
of heme binding to guanylate cyclase varies depending on the source of
the enzyme [27, 28]. The
literature contains no indication of the possible existence in tissues
of soluble guanylate cyclase in the heme-deficient form. We were the
first to demonstrate that rat platelet guanylate cyclase could not be
activated by sodium nitroprusside [29]. More
detailed investigation of this enzyme allowed us to conclude that,
contrary to the generally accepted notion, heme is not a constituent
part of the rat platelet guanylate cyclase molecule [30]. Therefore, rat platelets cannot be used as a
model to study the effect of NO and NO-generating compounds on platelet
guanylate cyclase. Naturally, the heme-deficiency of guanylate cyclase
impairs the endogenous regulation of the enzyme, decreases the efficacy
of nitrovasodilaters, and ultimately leads to disorders of vascular
tone.

This confronts us with the problem of measuring heme saturation of
guanylate cyclase. The problem was settled with the use of carnosine.
Carnosine (beta-alanyl-L-histidine) is a water-soluble
antioxidant, capable of forming chelate complexes. Because of its
antioxidant properties the substance is widely used in treatment of
inflammation, in wound healing, and cataract treatment [31, 32]. In studying the effect
of carnosine on human platelet guanylate cyclase we have first shown
[33] that carnosine, at a concentration producing
no effect on the basal activity of enzyme, strongly (by ~70%) inhibited
the stimulatory effect of sodium nitroprusside. However, carnosine did
not affect the slight activation by sodium nitroprusside of a
heme-deficient guanylate cyclase preparation, obtained by ion-exchange
chromatography [33]. Carnosine did not inhibit the
stimulatory effect of protoporphyrin IX on guanylate cyclase, which is
heme-independent [33]. Data obtained have
demonstrated that stimulation of guanylate cyclase by nitric oxide is a
combined result of: 1) a slight nonspecific and heme-independent
increase in activity (probably caused by oxidation of labile sulfhydryl
groups of the enzyme), and 2) stimulation of activity due to formation
of nitrosyl--heme complex. Carnosine inhibits the latter stimulation.
Since further experiments have shown that carnosine interacts with the
guanylate cyclase heme, it was postulated that carnosine specifically
inhibits heme-dependent NO-stimulation of guanylate cyclase and this
phenomenon can be used to estimate the degree of heme saturation of the
enzyme [33]. With the use of carnosine additional
data were obtained on the mechanism of vasodilatory action of sodium
nitroprusside and other nitroso complexes of some transition metals
differing in the character of NO oxidation. It is known that an
important factor in the vasodilatory action of sodium nitroprusside is
the degree of oxidation of the NO group and the readiness of its
release from the sodium nitroprusside molecule. It was shown earlier
that the NO group in sodium nitroprusside
Na2[FeNO+(CN)5] carries a positive
charge due to the nitrosonium cation [34]. Other
nitroso complexes of transition metals (structure analogs of sodium
nitroprusside) are poorly investigated. There is only one report [35] mentioning the absence of a pharmacological
(antihypertensive) effect of the anion
[Mn2+NO(CN)5]3- in which the NO group
is electrically neutral. Therefore, it seemed to us to be worthwhile to
study two analogs of sodium nitroprusside, the nitroso complexes of
transition metals (Cr and Co): K3[CrNO(CN)5] and
[CoNO(NH3)5]SO4, the NO groups of
which are neutral. Effects of these compounds on guanylate cyclase
activity from human platelets (heme-containing [36]) and rat platelets (heme-deficient [36]) were compared with the effect of sodium
nitroprusside on these same enzymes. As seen from the data presented in
the table, sodium nitroprusside is the most potent (16.2-fold)
activator of human platelet guanylate cyclase. NO activation is due to
the interaction between the sodium nitroprusside NO group and the
guanylate cyclase heme. This fact explains why sodium nitroprusside
cannot stimulate rat platelet guanylate cyclase, which is initially
heme-deficient. The table also shows that the Cr and Co nitroso
complexes only slightly stimulate guanylate cyclase activity, the
effects on the heme-containing (human platelet) and heme-deficient (rat
platelet) soluble guanylate cyclases are virtually equal in magnitude.
The data suggest that the mechanism of guanylate cyclase activation by
Cr and Co nitroso complexes is not heme-dependent. Further experiments
have confirmed this suggestion. It was shown [37]
that carnosine, a specific inhibitor of a heme-dependent stimulatory
effect on guanylate cyclase, reduces sodium nitroprusside-elicited
enzyme activation by 66 ± 4% without affecting the degree of the
enzyme activation by Cr and Co nitroso complexes [37]. In the case of heme-deficient human platelet
guanylate cyclase preparation obtained by ion-exchange chromatography,
its capacity to be activated by sodium nitroprusside was decreased by
90% but the magnitude of the stimulatory effects of Cr and Co nitroso
complexes remained virtually unchanged [37]. It
should be noted that nitroso complexes of Cr and Co (in contrast to
sodium nitroprusside) possessed no hypertensive action. Data obtained
allow us to conclude that in order for the hypotensive effect to be
materialized the activation of soluble guanylate cyclase must proceed
by the heme-dependent mechanism.

Effect of sodium nitroprusside (SNP),
K3[CrNO(CN)5] (I) and
[CoNO(NH3)5]SO4 (II) on guanylate
cyclase activity in 105,000g supernatants of human and rat
platelets
Note: The table shows the percent changes of guanylate cyclase
specific activities in the presence of SNP and compounds I or II. The
basal activity in the presence of Mg2+ was taken as 100%.

MECHANISM UNDERLYING THE ANTIAGGREGATORY ACTION OF NITRIC
OXIDE

It was known that nitric oxide and sodium nitroprusside both inhibit
platelet aggregation. This inhibitory effect is associated with the
ability of these compounds to activate soluble guanylate cyclase [38]. The antiaggregatory action of nitric oxide was
cleared up in detail after investigation of the role of guanylate
cyclase in regulation of platelet aggregation [39,
40]. When studying the dynamics of changes in
functioning of human platelet guanylate cyclase during ADP-induced
reversible aggregation in vitro, we found that immediately after
ADP addition the guanylate cyclase response to sodium nitroprusside
begins to increase with the concurrent increasing intraplatelet cGMP
level (Fig. 1, curves 1 and 2,
respectively). Curve 3 (Fig. 1) demonstrates
the effect of hemoglobin on guanylate cyclase activation by sodium
nitroprusside. It can be seen that in intact platelets, before ADP
addition hemoglobin increases the stimulatory effect of sodium
nitroprusside. This was interpreted as being due to the additional
formation of the nitrosyl--heme complex (at the expense of hemoglobin)
and its transfer to guanylate cyclase. Thus it would seem that in
natural conditions the guanylate cyclase is partly heme-deficient.
However, in the course of aggregation the stimulatory effect of
hemoglobin decreased and ultimately disappeared at the time point
corresponding to the maximum of the nitroprusside-induced activation of
the enzyme and to the highest cGMP level (see Fig. 1, curves 1-3). On reaching the
aggregation maximum (following by disaggregation) all the values
reverted to the baseline. Dynamics of the hemoglobin effect magnitude
on the nitroprusside activation (see Fig. 1, curve
3) reflects the gradually increasing heme saturation of the
enzyme throughout the aggregation process in accord with the enhanced
capacity of the enzyme to be activated by sodium nitroprusside. It
should be noted that dynamics of changes in guanylate cyclase
parameters is the same at all aggregation levels and is independent of
the degree of aggregation. However, the proportionality between the
time of achievement of the aggregation peak and the maximum of the
nitroprusside-produced activation was retained only with 45-50%
aggregation (see Fig. 2). In other words, these
changes occurred at the earliest stages of aggregation within the first
minutes of the aggregation process (see Fig. 2).
This is best demonstrated in the case of irreversible aggregation (Fig.
2d). At this stage the regulatory role of guanylate
cyclase is no longer revealed.

Fig. 1. Dynamics of changes in the cGMP level (1),
guanylate cyclase activation by sodium nitroprusside (2), and
guanylate cyclase heme saturation (3) at 45-50% reversible
platelet aggregation induced by 4-6 µM ADP. Abscissa, time (min)
after ADP addition. Ordinate, changes in the measured parameters (% of
initial value) (curves 1 and 2); changes in the effect of
hemoglobin on the nitroprusside-induced guanylate cyclase activation
(%) (curve 3). The mean values of five experiments of the same
type are given.

Fig. 2. Dynamics of changes in degree of guanylate cyclase
activation by sodium nitroprusside (1) at 20% (a), 50% (b), and
70% (c) reversible and 70% irreversible (d) ADP-induced human platelet
aggregation with platelet concentration 2.5·108 (a-c)
and 4.5·108 (d) per ml of plasma. Aggregograms
(curves 2). Ordinate, changes in the measured parameters (% of
the initial values). The results of a typical experiment are given.
Arrows indicate time of addition of ADP.

If sodium nitroprusside was introduced to platelets before the addition
of the aggregation inducer no increase in guanylate cyclase activation
was observed [41]. Thus, guanylate cyclase
provides a defense against aggregation. Moreover, sodium nitroprusside
not only prevents aggregation but also facilitates disaggregation.
Figure 3 shows that adding sodium nitroprusside at
the peak of aggregation initiates disaggregation, which explains the
inhibitory effect of nitroprusside on aggregation. Thus, guanylate
cyclase effects negative control over platelet aggregation: initiation
of aggregation is accompanied by increasing the enzyme activation and
accumulation of cGMP; the latter mediates a signal that induces
disaggregation. Therefore, the functioning of platelet guanylate
cyclase and the ability of platelets for aggregation are interrelated.
Indeed in comparing the functioning of platelet guanylate cyclase in
platelets of diabetes mellitus patients (types I and II) characterized
by increased aggregability and in normal platelets of healthy donors we
have found a decrease in the basal guanylate cyclase activity and a
reduced capacity of the enzyme to activation in platelets of diabetes
mellitus patients [42]. Moreover, the higher was
the platelet aggregability, the lower was the basal guanylate cyclase
activity and, accordingly, its capacity for activation [42]. It should be noted that the degree in guanylate
cyclase parameters was not associated with the aetiology of diabetes
mellitus but solely with hemostases system disturbances. Thus,
guanylate cyclase may be considered as a protective mechanism blocking
the development of aggregation. In this connection the directed
activation of guanylate cyclase by nitric oxide and NO-generating
compounds may be used to normalize pathologically increased platelet
aggregability. Since the regulatory role of cGMP is manifested at the
earliest stages of aggregation (see Fig. 2) new
activators of guanylate cyclase will be capable not only to reduce
platelet aggregability but also prevent their spontaneous aggregation
and hence the appearance and development of vascular complications.

Fig. 3. Effect of sodium nitroprusside (0 (1),
10-4 (2), 10-5 (3), 10-6
(4), 10-7 M (5)) on ADP-induced human platelet
aggregation. Abscissa, time (min) after ADP addition; ordinate,
platelet aggregation (%). The arrows indicate the time of addition of
ADP and sodium nitroprusside (SNP). The results of five experiments of
the same type are presented.

Clearly, the molecular mechanism of antiaggregatory action of nitric
oxide and NO donors is connected with activation of soluble guanylate
cyclase and accumulation of cGMP. However, the molecular mechanism of
cGMP participation in the aggregation process is yet incompletely
understood.

Figure 4 represents our hypothetical scheme of
likely sites of action of cGMP as a regulator of platelet aggregation.
Figure 4 shows that cGMP inhibits the liberation of
arachidonic acid, thus preventing the activation of phospholipase
A2; however, cGMP does not affect the subsequent steps of
arachidonic acid breakdown mediated by cyclooxygenase and thromboxane
synthetase, both of which stimulate the accumulation of Ca2+
and cause platelet activation and aggregation. cGMP impedes the
formation of 1,2-diacylglycerol and inositol trisphosphate, through the
inhibition of phospholipase C activation. 1,2-Diacylglycerol is a
potent activator of protein kinase C which phosphorylates platelet
proteins (20 and 40 kD), causes platelet activation and aggregation.
cGMP prevents the formation of inositol trisphosphate, suppresses the
accumulation of Ca2+ thereby inhibiting the platelet
activation and aggregation. cGMP also inhibits the formation of
phosphatidic acid, which is readily formed in platelets from
1,2-diacylglycerol under the action of 1,2-diacylglycerol
phospholipase; phosphatidic acid acts as an ionophore, facilitating the
release of intracellular Ca2+ and causing activation and
aggregation of platelets. In other words, cGMP prevents phospholipid
breakdown (including inositol phospholipids) and inhibits platelet
aggregation through a common mechanism inhibiting Ca2+
accumulation [43].

Fig. 4. Model summarizing the possible role of platelet guanylate
cyclase (GC) and cGMP in regulation of platelet
aggregation.

Thus, antihypertensive and antiaggregatory properties of nitric oxide
are due to soluble guanylate cyclase activation and accumulation of
cGMP.

In connection with the above-reviewed data, the synthesis of new NO
donors and revealing among them possible guanylate cyclase activators
seems to be promising and pertinent for solution of one the most
fundamental problems of modern biological and medical chemistry, the
directed search for and synthesis of new effective antihypertensive and
antiaggregatory preparations based on the investigation of the effects
of NO-generating compounds on soluble guanylate cyclase.

Thus, we have first studied a new class of newly synthesized compounds,
derivatives of 1,2-diazetine-1,2-di-N-oxides capable of non-enzymatic
generation of nitric oxide by a principally new mechanism of nitric
oxide splitting at physiological pH values and without the
participation of thiols [44, 45] according to Eq. (1):

Four of the seven derivatives tested exhibited a distinct correlation
between the ability of being decomposed with nitric oxide formation,
activation of soluble guanylate cyclase [45],
inhibition of platelet aggregation, and acceleration of platelet
disaggregation [46]. Furthermore, studies of the
spasmolytic activity of compounds on isolated rings of aorta and their
hypotensive effect on urethane-narcoticized spontaneously hypertensive
rats (carried out at the Chemical Pharmaceutical Research Institute,
Moscow) have shown that with each of the four derivatives of
diazetine-di-N-oxides there is a full correlation between the ability
of being decomposed with NO formation, soluble guanylate cyclase
activation, and manifestation of the above-mentioned physiological
effects [47]. Among the compounds tested,
3-brom-4-methyl-3,4-tetramethylene-diazetine-di-N-oxide has proved to
be most effective, its spasmolytic effect being commensurate with
glycerotrinitrate activity [47].

The next class of new guanylate cyclase activators capable of nitric
oxide generation were derivatives of guanidine thiols. Guanidine thiols
contain both the guanidine and SH groups which act, respectively, as
donor and acceptor of nitric oxide. It was shown that
beta-mercaptoethylguanidine
(HS-CH2-CH2-NH-C=NH(NH2)) proved to be
a much more potent activator of soluble guanylate cyclase than was
L-arginine [48]. In our view, this is explained by
formation of unstable intramolecular nitrosothiols promoting NO
transfer to the guanylate cyclase heme and hence the enzyme activation.
To ascertain the role of guanidine thiols SH groups in increasing their
stimulatory effect on guanylate cyclase, three compounds were tested:
beta-mercaptoethylguanidine (MEG), mercaptoethylguanidine
disulfide (MEG-disulfide), and the SH-group methylated MEG derivative
S-methyl-mercaptoethylguanidine (S-methyl-MEG) with the following
formula:

All these compounds proved to be guanylate cyclase activators. The
stimulatory effect of these compounds was blocked by
NG-monomethyl-L-arginine, a well-known inhibitor of
NO-synthase [49]. The degree of guanylate cyclase
activation by MEG and MEG-disulfide was, respectively, two and four
times higher than that of L-arginine [49]. The
stimulatory effect of S-methyl-MEG was of the same order as that of
L-arginine [49]. Thus, the important role of
S-acceptor group of guanidine thiols in the intensification of
guanylate cyclase activation was demonstrated providing a plausible
explanation for different intensities of guanylate cyclase activation
by the compounds tested [49]. In full accordance
with the intensity of the stimulatory effect of these compounds on
guanylate cyclase activity are their antiaggregatory properties [50] and their hypotensive effect on intravenous
injection to spontaneously hypertensive rats [51].

Of interest is the dependence between the chemical structure of a
compound as well as its ability to generate NO and to activate human
platelet guanylate cyclase revealed in studies yet another class of
compounds--derivatives of oximes of quinuclidin-3-one--generating NO by
oxidation [52].

A group of compounds with different stereochemistry identified on the
basis of H1-NMR-spectroscopy was obtained [52]. It was shown that the most active compound in
this line, based on electrochemical data and on stimulatory effect on
guanylate cyclase, is the compound containing the ortho-hydroxy group
in the aryl ring. Consequently, the oxime and phenolic fragments are in
near proximity to each other; it may be suggested, therefore, that the
phenolic hydroxy group possesses NO acceptor properties allowing it to
promote NO transfer onto guanylate cyclase heme and consequently, the
activation of the enzyme. The methylation of phenolic hydroxyl blocks
its NO acceptor properties and decrease its stimulatory effect on
guanylate cyclase [52]. An analogous dependence of
the guanylate cyclase activation intensity on the presence of free
phenolic hydroxyl in the oxime molecule was revealed with some other
oximes--derivatives of p-hydroxy- and
p-methoxybenzaldehydes. The former compound
(OH-C6H4-CH=NOH) (0.1 mM) was able to activate
soluble guanylate cyclase (3.5 ± 0.41)-fold, the latter
(H3C-O-C6H4-CH=NOH) (0.1 mM), (1.4
± 0.17)-fold. Thus, the acceptor properties of phenolic
hydroxyls were first demonstrated.

Study of the dependence of the degree of guanylate cyclase activation on
the structure of NO generating compounds has shown that directed
introduction of NO acceptor groups (such as SH groups and phenolic
hydroxyl) into molecules of new NO donors increases the stimulatory
effect of these compounds on the enzyme activity.

Thus, synthesis of new NO donors and study of their effects on soluble
guanylate cyclase--to reveal among them the most active enzyme
stimulators for pharmacological uses--serve as a molecular basis for
directed search for and creation of new effective antihypertensive and
antiaggregatory preparations.

Indeed, in studying a great number of newly synthesized NO donors
belonging to different classes of compounds we have clearly
demonstrated that, inside each class, the more active was a stimulator
of guanylate cyclase activity the more pronounced were its
antihypertensive and antiaggregatory effects. It appears that, based on
the intensity of guanylate cyclase stimulation by a given compound, the
pharmacological efficiency of each particular newly synthesized NO
donor may be predicted.

This work received financial support from the Russian Foundation for
Basic Research.